30 research outputs found
Recommended from our members
Engineering Yarrowia lipolytica for high lipid production
Among potential value-added fuels and chemicals, fatty acid-based chemicals are important due to their wide use in industrial processes and in daily life. Fatty acids produced from microbial systems could provide a sustainable supply to replace the current costly and unsustainable process using plant oil or animal fat. The oleaginous yeast Yarrowia lipolytica naturally possesses moderate lipid production capacity and grows on different kinds of biomass and organic waste. However, fatty acid production from native, un-engineered strains is not economically viable. Therefore, this work develops strategies inspired from synthetic biology and metabolic engineering to expand the engineering potential of Y. lipolytica — helping to establish this organism as a premier platform for industrial-level, high lipid production as well as providing a platform for uncovering novel understanding of lipogenesis.
To do so, first, novel synthetic promoters and high expression plasmid were necessary to achieve the ability to tune gene expression levels inside the cell. We developed a hybrid promoter engineering strategy to create a promoter library exhibiting a range of more than 400-fold in terms of mRNA levels as well as engineered plasmids with regulated centromeric function to achieve a 2.7 fold expression range. Next, a rational and evolutionary metabolic engineering approach was coupled with genomic and transcriptomic studies to both engineer and understand underlying lipogenesis in this organism. Through the engineering efforts, we successfully increased the lipid production titer to over 40 g/L in bioreactor as well as identified novel lipogenic enhancers and mechanisms. In addition, we identified and characterized a mutant mga2 protein with superior lipogenesis enhancing capacity, which can regulate fatty acid desaturation and carbon flux inside the cells. Collectively, these studies have facilitated the utilization of Y. lipolytica as an industrially relevant microbial lipid production platform and supplied novel understanding of its lipogenesis process. The methods and concepts developed here can also be adapted to other oleaginous microbes and serve as a template for enabling value-added chemical production in other nonconventional organism.Biochemistr
Combined aptamer and transcriptome sequencing of single cells.
The transcriptome and proteome encode distinct information that is important for characterizing heterogeneous biological systems. We demonstrate a method to simultaneously characterize the transcriptomes and proteomes of single cells at high throughput using aptamer probes and droplet-based single cell sequencing. With our method, we differentiate distinct cell types based on aptamer surface binding and gene expression patterns. Aptamers provide advantages over antibodies for single cell protein characterization, including rapid, in vitro, and high-purity generation via SELEX, and the ability to amplify and detect them with PCR and sequencing
Recommended from our members
Linked optical and gene expression profiling of single cells at high-throughput.
Single-cell RNA sequencing has emerged as a powerful tool for characterizing cells, but not all phenotypes of interest can be observed through changes in gene expression. Linking sequencing with optical analysis has provided insight into the molecular basis of cellular function, but current approaches have limited throughput. Here, we present a high-throughput platform for linked optical and gene expression profiling of single cells. We demonstrate accurate fluorescence and gene expression measurements on thousands of cells in a single experiment. We use the platform to characterize DNA and RNA changes through the cell cycle and correlate antibody fluorescence with gene expression. The platform's ability to isolate rare cell subsets and perform multiple measurements, including fluorescence and sequencing-based analysis, holds potential for scalable multi-modal single-cell analysis
Recommended from our members
Compositions and methods for lipid production
Described herein, inter alia, are compositions, oleagnious organisms, and methods useful for producing lipids, lipid precursors, and/or oleochemicals.Board of Regents, University of Texas Syste
Recommended from our members
RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes.
Synthetic biology and metabolic engineering seek to re-engineer microbes into living foundries for the production of high value chemicals. Through a design-build-test cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening. Well plate assays are flexible but with limited throughput, whereas droplet microfluidic techniques are ultrahigh-throughput but require a custom assay for each target. Here we present RNA-aptamers-in-droplets (RAPID), a method that greatly expands the generality of ultrahigh-throughput microfluidic screening. Using aptamers, we transduce extracellular product titer into fluorescence, allowing ultrahigh-throughput screening of millions of variants. We demonstrate the RAPID approach by enhancing production of tyrosine and secretion of a recombinant protein in Saccharomyces cerevisiae by up to 28- and 3-fold, respectively. Aptamers-in-droplets affords a general approach for evolving microbes to synthesize and secrete value-added chemicals.Screening libraries of genetically engineered microbes for secreted products is limited by the available assay throughput. Here the authors combine aptamer-based fluorescent detection with droplet microfluidics to achieve high throughput screening of yeast strains engineered for enhanced tyrosine or streptavidin production
Recommended from our members
Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage.
Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1+SiglecF+ transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo. Human orthologs of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury
Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells.
We show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes' expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC
Human Pluripotent Stem Cell-Derived Cardiac Tissue-like Constructs for Repairing the Infarcted Myocardium.
High-purity cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are promising for drug development and myocardial regeneration. However, most hiPSC-derived CMs morphologically and functionally resemble immature rather than adult CMs, which could hamper their application. Here, we obtained high-quality cardiac tissue-like constructs (CTLCs) by cultivating hiPSC-CMs on low-thickness aligned nanofibers made of biodegradable poly(D,L-lactic-co-glycolic acid) polymer. We show that multilayered and elongated CMs could be organized at high density along aligned nanofibers in a simple one-step seeding process, resulting in upregulated cardiac biomarkers and enhanced cardiac functions. When used for drug assessment, CTLCs were much more robust than the 2D conventional control. We also demonstrated the potential of CTLCs for modeling engraftments in vitro and treating myocardial infarction in vivo. Thus, we established a handy framework for cardiac tissue engineering, which holds high potential for pharmaceutical and clinical applications
Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach▿†
The development of strong and tunable promoter elements is necessary to enable metabolic and pathway engineering applications for any host organism. Here, we have expanded and generalized a hybrid promoter approach to produce libraries of high-expressing, tunable promoters in the nonconventional yeast Yarrowia lipolytica. These synthetic promoters are comprised of two modular components: the enhancer element and the core promoter element. By exploiting this basic promoter architecture, we have overcome native expression limitations and provided a strategy for both increasing the native promoter capacity and producing libraries for tunable gene expression in a cellular system with ill-defined genetic tools. In doing so, this work has created the strongest promoters ever reported for Y. lipolytica. Furthermore, we have characterized these promoters at the single-cell level through the use of a developed fluorescence-based assay as well as at the transcriptional and whole-cell levels. The resulting promoter libraries exhibited a range of more than 400-fold in terms of mRNA levels, and the strongest promoters in this set had 8-fold-higher fluorescence levels than those of typically used endogenous promoters. These results suggest that promoters in Y. lipolytica are enhancer limited and that this limitation can be partially or fully alleviated through the addition of tandem copies of upstream activation sequences (UASs). Finally, this work illustrates that tandem copies of UAS regions can serve as synthetic transcriptional amplifiers that may be generically used to increase the expression levels of promoters
Accurate Bulk Quantitation of Droplet Digital Polymerase Chain Reaction.
Droplet digital PCR provides superior accuracy for nucleic acid quantitation. The requirement of microfluidics to generate and analyze the emulsions, however, is a barrier to its adoption, particularly in low resource settings or clinical laboratories. Here, we report a novel method to prepare ddPCR droplets by vortexing and readout of the results by bulk analysis of recovered amplicons. We demonstrate the approach by accurately quantitating SARS-CoV-2 sequences using entirely bulk processing and no microfluidics. Our approach for quantitating reactions should extend to all digital assays that generate amplicons, including digital PCR and LAMP conducted in droplets, microchambers, or nanoliter wells. More broadly, our approach combines important attributes of ddPCR, including enhanced accuracy and robustness to inhibition, with the high-volume sample processing ability of quantitative PCR